A method as set forth in the opening paragraph is known from U.S. Pat. No. 5,412,210, and makes use of the insight that changing the primary beam energy in SEM leads to deeper penetration inside the sample being investigated. In principle, such an approach can be used to generate three-dimensional (3D) tomograms of regions of interest in the sample. Up to now, attempts to exploit this approach have involved acquiring two or more images with increasing primary beam energy, adjusting contrast between the images, and then subtracting lower-energy images from higher-energy images to reveal submerged layers in the sample.
A drawback of such known approaches is that said inter-image contrast adjustment (which is a key step) can only be performed using knowledge about the composition and geometry of the sample. Consequently, prior applications of this technique have tended to limit themselves to wafer defect inspection and other semiconductor applications, in which there is generally good a priori knowledge of the sample's (default) composition and geometry. Since the required compositional and geometrical information is typically not available for biological samples, the known technique has not yet been successfully applied to investigations in the life sciences.